In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of the rat mucosal mast cell tumor line RBL after transfection with genes for cytotoxic lymphocyte granule components. We have analyzed the cytotoxic activity of triple, double, and single RBL transfectant clones expressing cytolysin (cy) and the granule serine proteases granzyme A (gza) and granzyme B (gzb). With red cell targets, granzyme expression has no effect on lytic potency, which is higher than cloned CTL for all RBL-cy-x clones studied. On tumor targets, RBL-cy clones show only modest cytotoxicity, with no accompanying target nuclear damage. With four different tumor targets, both lytic potency and target DNA damage of RBL-cy-gzb and RBL-cy-gza-gzb clones correlated with granzyme expression but not cytolysin expression. RBL-cy-gza-gzb clones show lytic and nucleolytic activities close (3x-4x fewer lytic units) to cloned CTL. Using two different tumor target cells, comparison of the nucleolytic and cytolytic activities of both RBL-cy-gza RBL-cy-gzb clones with RBL-cy-gza-gzb clones reveals that the latter are much more active than the sum of the activities of the double transfectants, showing that the two granzymes interact synergistically to trigger target cell death and nuclear breakdown. To see if the nuclear damage is required for the granzyme-mediated cell death pathway, 51Cr release from enucleated tumor target cells was compared to intact tumor cells and red cells. The enucleated cytoplast targets behaved like the intact tumor cells and not red cells, in that RBL-cy-gza-gzb killed them much better than RBL-cy. Similar results were obtained using Fas-bearing target cells and the Fas ligand-bearing effector cell line d11S. Thus we conclude that the nucleus is not involved in either the granzyme-triggered death pathway or in the Fas-triggered death pathway, which are the two principle death pathways triggered by cytotoxic lymphocytes.